Myocardial Injury Associated with Mitochondria-Derived Oxygen Free Radical(s)

描述（由申请人提供）：线粒体作为能量产生的主要来源，是正常细胞功能所必需的。有相当多的证据支持线粒体功能障碍在心肌梗死和心力衰竭等心脏病中的关键作用。缺血后心肌水平的能量代谢缺陷与线粒体中氧自由基和NO的过量产生有关。在心肌缺血再灌注损伤过程中，线粒体复合体I （NQR）、复合体II （SQR）和其他ETC蛋白中检测到蛋白s -谷胱甘肽化（PrSSG）和蛋白硝化作用的改变。NQR/ sqr衍生的氧化修饰的改变与线粒体中氧自由基的产生、NO代谢和氧化还原硫醇的稳态密切相关。NQR或SQR中活性和不稳定半胱氨酸残基的s -谷胱甘肽化是可逆修饰，而活性半胱氨酸残基的s -磺化是不可逆修饰。我们的主要假设是，这两种半胱氨酸修饰都受到缺血后心脏线粒体氧化还原状态的高度调节，而线粒体氧化还原状态受ROS产生、NO代谢和谷胱甘肽池的稳态控制。本研究的长期目标是阐明线粒体氧化还原信号介导心肌损伤的分子机制，了解其发病机制，并开发心血管疾病的治疗方法。我们将通过使用新型动物模型、EPR光谱法和质谱法，对线粒体中导致蛋白质s -谷胱甘肽化/磺化的主要信号通路的关键假设进行验证。特异性目的1将确定缺血后心肌线粒体中是否会诱导NQR和SQR的不可逆蛋白S-磺化。心肌梗死后NQR和SQR标记的蛋白磺化将用质谱法进行表征。将产生一个序列特异性的磺化抗体来检测体外和体内的这一事件。EPR光谱与自旋探针和自旋陷阱将被用来测量线粒体的氧化还原状态和“氧生成活性”从缺血后心脏分离。具体目标2

DESCRIPTION (provided by applicant): Mitochondria as the major source of energy generation are essential for proper cellular function. There is considerable evidence supporting the key role of mitochondrial dysfunction in heart disease such as myocardial infarction and heart failure. At the myocardial level of the post-ischemic heart, a defect in energy metabolism associated with overproducing oxygen free radicals and NO in mitochondria was marked. Alterations of protein S-glutathionylation (PrSSG) and protein nitration have been detected in the mitochondrial complex I (NQR), complex II (SQR), and other ETC proteins during myocardial ischemia and reperfusion injury. Alterations of NQR/SQR-derived oxidative modifications are closely linked to oxygen free radical production, NO metabolism, and homeostasis of redox thiols in mitochondria. S-glutathionylation of the reactive and labile cysteine residues in the NQR or SQR is a reversible modification, whereas S-sulfonation of reactive cysteine residues is an irreversible modification. Our central hypotheses are that both cysteinyl modifications are highly regulated by the redox status in the mitochondria of the post-ischemic heart, and the mitochondrial redox status is controlled by ROS production, NO metabolism, and the homeostasis of the GSH pool. The long term objectives of this research are to elucidate the molecular mechanism of mitochondrial redox signals in the mediation of myocardial injury, to understand the pathogenesis, and to develop a treatment for cardiovascular diseases. The key hypotheses of the major signal pathway leading to protein S-glutathionylation/sulfonation in mitochondria will be tested by pursuing the following specific aims using novel animal models, EPR spectrometry, and mass spectrometry. Specific aim 1 will determine whether irreversible protein S- sulfonation of NQR and SQR is induced in the mitochondria of the post-ischemic myocardium. The protein sulfonation marked in the NQR and SQR after myocardial infarction will be characterized by mass spectrometry. A sequence-specific antibody for sulfonation will be generated to detect this event in vitro and in vivo. EPR spectrometry with a spin probe and a spin trap will be used to measure the redox status and "O2- generation activity of mitochondria isolated from the post-ischemic heart. Specific aim 2 will determine the role of glutathione reductase (GR) in the mechanism of glutathionylation of NQR/SQR and regulation of overall mitochondrial function in the post-ischemic heart. We will use a pharmacologic approach and mice deficient in GR (gsr-/-) to determine whether (i) enhancing GSSG in vivo will increase glutathionylation of NQR and SQR, and (ii) whether or not increasing NQR/SQR glutathionylation in vivo will be protective and reduce the susceptibility of mitochondria to post-ischemic injury. Specific aim 3 will ascertain the role of eNOS in the mechanism of NQR/SQR glutathionylation and regulation of overall mitochondrial function in the post-ischemic heart. Mice with an eNOS-/- and a cardiac-specific eNOS-/- genotype will serve as an excellent in vivo model for studying the regulation of NQR/SQR glutathionylation, mitochondrial redox status, and its "O2- generation activity via NO metabolism. We will also create a novel mouse model by crossing cardiac-specific SOD2 transgenic mice with eNOS-/- mice in order to determine whether increased SOD2 signaling in mitochondria is sufficient to correct oxidative injury resulting from eNOS deficiency and post-ischemic injury.